Abstract Pancreatic ductal adenocarcinoma (PDAC) is a deadly disease with a 5-year survival rate of only 8%. Despite success in other cancer types, immunotherapy approaches in PDAC have not shown efficacy. PDAC demonstrates a heterogeneous and immunosuppressive tumor microenvironment (TME) that is poorly understood and serves as a barrier to effective immunotherapy strategies in this disease. We propose that an improved understanding of the TME and novel approaches that target key tumor-stroma interactions will enable remodeling of the immunosuppressive TME to enhance the efficacy of current and future immunotherapy strategies. In particular, we believe that successful combination immunotherapy approaches in PDAC will include strategies that alter myeloid cells to relieve immunosuppression, cytotoxic therapies that target tumor cells to improve immune response, and agents that augment anti-tumor T cell activity. In this project, we will perform a comprehensive characterization of the PDAC TME in both primary and metastatic PDAC in the baseline untreated context as well as across multiple different clinical therapies. In Aim 1, we will utilize single-cell transcriptomic and proteomic technologies to provide a cellular atlas of the PDAC TME at unprecedented resolution. In Aim 2, we will examine how the PDAC TME changes with chemotherapy, radiation therapy and a novel CCR2 inhibitor that modulates macrophage recruitment in the TME. For these studies, we will utilize human samples derived from both resectable and metastatic patients on clinical trials at Dana-Farber Cancer Institute. We will employ a novel ex vivo co-culture approach to enable rapid functional evaluation of tumor-stroma interactions and how they may impact immunotherapy responses. Lastly, in Aim 3 we will employ faithful immune competent PDAC mouse models and a novel cytokine delivery platform to investigate how targeted cytokine delivery to the TME may alter myeloid cell recruitment and function and improve immune responses. We have assembled a multi-disciplinary collaborative team including experts in PDAC biology and genetics, immunologists and translational oncologists to comprehensively study PDAC TME and identify novel opportunities to develop combination immunotherapy approaches in this devastating disease.